CN113683706B

CN113683706B - Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea

Info

Publication number: CN113683706B
Application number: CN202111068018.1A
Authority: CN
Inventors: 王海波; 刁鹏飞; 谭双; 邓红娟; 李洪凌; 马学东; 曹旗; 任洪林
Original assignee: Weifang Xiashan Weitai Biotechnology Co ltd
Current assignee: Weifang Xiashan Weitai Biotechnology Co ltd
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2023-02-28
Anticipated expiration: 2041-09-13
Also published as: CN113683706A

Abstract

The invention provides a construction method, an expression system and application of a multiple fusion recombinant protein capable of preventing piglet diarrhea, and relates to the technical field of biological medicines. According to the invention, by cloning heat-resistant enterotoxin and heat-labile enterotoxin genes of ETEC, and alpha toxin and beta 1 toxin genes of clostridium welchii, functional regions of partial genes are connected in series by using a SOE-PCR method, an LT I-LT II-ST-Cp recombinant sequence is amplified, an expression vector is successfully constructed, and a recombinant protein LT I-LT II-ST-Cp is successfully expressed. The recombinant protein LT I-LT II-ST-Cp has good specificity of each segment, no toxicity after immunization of mice, high antibody titer and good protection effect on diarrhea. The vaccine prepared by the invention has a protection rate of more than 80% on red dysentery and yellow dysentery of piglets, and has good effect and strong protection.

Description

Construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a construction method, an expression system and application of a multi-union fusion recombinant protein capable of preventing piglet diarrhea.

Background

Piglet diarrhea is a typical high-incidence disease under intensive pig raising production conditions. The disease is one of the most serious piglet disease groups at present and is also an important cause of piglet death. According to investigation, the death of piglets due to diarrhea can account for 39.8 percent of the total number of the death of piglets. Therefore, how to take effective measures to prevent and control the diarrhea of the piglets and improve the survival rate of the piglets becomes an important topic for the production of the pig industry.

Research shows that one of the main causes causing the diarrhea of piglets is infectious bacterial diarrhea, and colibacillosis of piglets is a type of infectious disease caused by pathogenic Escherichia coli, wherein enterotoxigenic Escherichia coli (ETEC) is an important pathogenic bacterium causing the diarrhea of piglets; in addition, the piglet red diarrhea caused by the clostridium welchii is also important infectious bacterial diarrhea of piglet diarrhea, but at present, a vaccine capable of simultaneously preventing and controlling a plurality of pathogenic bacteria causing piglet diarrhea does not exist.

Disclosure of Invention

In view of the above, the invention aims to provide a construction method of a multiple fusion recombinant protein capable of preventing piglet diarrhea, an expression system and application thereof, which have good immunity and strong specificity and provide effective vaccine protection for preventing piglet diarrhea.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a concatameric fusion recombinant protein LT I-LT II-ST-Cp, the amino acid sequence of which is shown in SEQ ID NO. 1.

Preferably, the nucleotide sequence for coding the recombinant protein LT I-LT II-ST-Cp is shown as SEQ ID NO. 2.

The invention also provides a construction method of the coding gene of the recombinant protein LT I-LT II-ST-Cp, which comprises the following steps: the encoding gene is obtained by connecting a partial functional region of a heat-resistant enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of a thermolabile enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of an alpha toxin gene derived from clostridium welchii and a partial functional region of a beta 1 toxin gene derived from clostridium welchii in series by using an SOE-PCR method.

Preferably, the partial functional region of the heat-labile enterotoxin gene derived from enterotoxigenic escherichia coli includes: LT Ia, LT Ib, LT IIAa, LT II Ab, LT II ca and LT II cb; the CDS sequence of LT Ia is shown as 1-258bp of SEQ ID NO.2, the CDS sequence of LT Ib is shown as 259-381bp of SEQ ID NO.2, the CDS sequence of LT II Aa is shown as 382-627bp of SEQ ID NO.2, the CDS sequence of LT II Ab is shown as 628-753bp of SEQ ID NO.2, the CDS sequence of LT II ca is shown as 754-846bp of SEQ ID NO.2, and the CDS sequence of LT II cb is shown as 847-927bp of SEQ ID NO. 2;

the partial functional regions of the heat-resistant enterotoxin gene derived from enterotoxin-producing escherichia coli comprise: ST a and ST b; the CDS sequence of ST a is shown as 928-1044bp of SEQ ID NO.2, and the CDS sequence of ST b is shown as 1045-1143bp of SEQ ID NO. 2;

part of the functional region of the clostridium welchii-derived alpha toxin gene includes Cp α; the CDS sequence of the Cp alpha is shown as 1144-1266bp of SEQ ID NO. 2;

a part of the functional region of the clostridium welchii-derived β 1 toxin gene includes Cp β; the CDS sequence of Cp beta is disclosed as 1267-1524bp of SEQ ID NO. 2.

The invention also provides a recombinant expression system of the recombinant protein LT I-LT II-ST-Cp.

Preferably, the recombinant expression system comprises a eukaryotic expression system or a prokaryotic expression system.

The invention also provides a recombinant expression vector for expressing the recombinant protein LT I-LT II-ST-Cp, wherein the basic expression vector of the recombinant expression vector comprises pET-28a, and the nucleotide sequence for coding the recombinant protein LT I-LT II-ST-Cp is inserted between Nco I enzyme cutting sites and Xho I enzyme cutting sites of pET-28 a.

The invention also provides an inducible expression method of the recombinant protein LT I-LT II-ST-Cp, which comprises the following steps: transferring the recombinant expression vector into an escherichia coli competent cell E.coli DH5 alpha, extracting a plasmid, then transforming the escherichia coli competent cell E.coli BL21 (DE 3), and inducing by IPTG to obtain the recombinant protein LT I-LT II-ST-Cp.

The invention also provides application of the recombinant protein LT I-LT II-ST-Cp or the recombinant protein LT I-LT II-ST-Cp obtained by the induction expression method in preparation of a piglet diarrhea concatenated vaccine.

The invention also provides a concatenated subunit genetic engineering vaccine for piglet diarrhea, which comprises the recombinant protein LT I-LT II-ST-Cp or the recombinant protein LT I-LT II-ST-Cp obtained by utilizing the induction expression method and an immunologic adjuvant.

Has the advantages that: according to the invention, by cloning heat-resistant enterotoxin and heat-labile enterotoxin genes of ETEC, and alpha toxin and beta 1 toxin genes of clostridium welchii, functional regions of partial genes are connected in series by using a SOE-PCR method, an LT I-LT II-ST-Cp recombinant sequence is amplified, an expression vector pET-28a-LT I-LT II-ST-Cp is successfully constructed, and a recombinant protein LT I-LT II-ST-Cp is successfully expressed by using an escherichia coli prokaryotic expression system. The biological activity of the recombinant protein LT I-LT II-ST-Cp is verified by animal experiment analysis, the recombinant protein LT I-LT II-ST-Cp has good specificity of each segment, no toxicity after mice are immunized, the antibody titer is higher, the antiserum has good specificity to ETEC natural enterotoxin protein and clostridium welchii natural alpha and beta 1 toxin, and an immune efficacy detection experiment shows that the recombinant protein LT I-LT II-ST-Cp has good protection effect on diarrhea. The invention also utilizes the protein denaturation technology to inactivate the toxicity of the genetic engineering toxin, retains the immunogenicity similar to that of the natural toxin, and is assisted with proper immunologic adjuvant to prepare the piglet diarrhea concatenated recombinant genetic engineering vaccine, and the vaccine has the protection rate of more than 80 percent on piglet red dysentery and piglet yellow dysentery, good effect and strong protection.

Drawings

FIG. 1 shows the PCR amplification results of the target gene of LT I-LT II-ST-Cp, wherein lane 1 shows DL 2000DNA Marker; lane 2 shows the LT I-LT II-ST-Cp target fragment;

FIG. 2 is an SDS-PAGE expression analysis of LT I-LT II-ST-Cp recombinant protein; wherein lane 1 represents the standard protein molecular weight; lane 2 shows a negative control with no inducer added; lanes 3-6 show incubation for 8h after IPTG was added;

FIG. 3 shows the purification of recombinant proteins; wherein lane 1 represents the standard protein molecular weight; lane 2 shows that the recombinant LT I-LT II-ST-Cp protein is not induced; lane 3 shows the induction of LT I-LT II-ST-Cp recombinant protein for 8h; lane 4 shows purified LT I-LT II-ST-Cp recombinant protein;

FIG. 4 shows the specificity of the recombinant protein, and FIG. 1 shows the anti-LT Ia antibody assay; 2, verifying an anti-LT Ib antibody; 3, verifying an anti-LT II Aa antibody; 4, verifying an anti-LT II Ab antibody; 5, verifying an anti-LT II ca antibody; 6 is anti-LT IIcb antibody verification; 7, anti-ST a antibody validation; 8, anti-ST b antibody validation; 9 anti-Cp α antibody validation; verification by an anti-Cp beta 1 antibody is 10;

FIG. 5 is a graph showing the level of serum antibody changes at different periods of time after immunization;

figure 6 demonstrates antiserum specificity for ETEC;

FIG. 7 shows the verification of antiserum specificity by Clostridium welchii.

Detailed Description

The nucleotide sequence of the recombinant protein LT I-LT II-ST-Cp coded by the invention is preferably shown as SEQ ID NO. 2.

The invention also provides a construction method of the coding gene of the recombinant protein LT I-LT II-ST-Cp, which comprises the following steps: and (3) connecting a partial functional region of a heat-resistant enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of a thermolabile enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of an alpha toxin gene derived from clostridium welchii and a partial functional region of a beta 1 toxin gene derived from clostridium welchii in series by using an SOE-PCR method to obtain the coding gene.

The functional regions of the thermolabile enterotoxin gene derived from enterotoxin-producing Escherichia coli according to the present invention preferably include: LT Ia, LT Ib, LT IIAa, LT II Ab, LT II ca and LT II cb; the partial functional region of the heat-resistant enterotoxin gene derived from enterotoxin-producing escherichia coli preferably includes: ST a and ST b; preferably, the partial functional region of the clostridium welchii-derived alpha toxin gene comprises Cp α; preferably, a part of the functional region of the clostridium welchii-derived β 1 toxin gene includes Cp β.

The CDS sequence of the LT Ia is preferably 373-630 bp of the CDS sequence of the gene with the accession number of BAA25725.1, the CDS sequence of the LT Ia is preferably shown as SEQ ID NO.21-258bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.11-86 aa.

The CDS sequence of the LT Ib is preferably 223-346 bp of the CDS sequence of a gene with the accession number of BAA25726.1, the CDS sequence of the LT Ib is preferably shown as SEQ ID NO.2259-381bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.187-127 aa.

The CDS sequence of the LT II Aa is preferably 544-789 bp of the CDS sequence of the gene with the accession number WP _095374651.1, the CDS sequence of the LT II Aa is preferably shown as SEQ ID NO.2382-627bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1128-209 Aa.

The CDS sequence of the LT II Ab is preferably 214-339 bp of the CDS sequence of the gene with the accession number WP _095389218.1, the CDS sequence of the LT II Ab is preferably shown as SEQ ID NO.2628-753bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1210-251 aa.

The CDS sequence of the LT II ca is preferably 58-150 bp of the CDS sequence of the gene with the accession number of ALO79807.1, the CDS sequence of the LT II ca is preferably shown as SEQ ID NO.2754-846bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1252-282 aa.

The CDS sequence of the LT IIcb is preferably 190-270 bp of the CDS sequence of the gene with the accession number of ALO79813.1, the CDS sequence of the LT IIcb is preferably shown as SEQ ID NO.2847-927bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1283-309 aa.

The CDS sequence of the ST a is preferably 58-174 bp of the CDS sequence of the gene with the accession number of CAD87828.1, the CDS sequence of the ST a is preferably shown as SEQ ID NO.2928-1044bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1310-348 aa.

The CDS sequence of ST b is preferably 70-168 bp of the CDS sequence of the gene with the accession number of CAD87835.1, the CDS sequence of ST b is preferably shown as SEQ ID NO.21045-1143bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1349-381 aa.

The CDS sequence of the Cp alpha is preferably 148-270 bp of the CDS sequence of the gene with the accession number of AQN80672.1, the CDS sequence of the Cp alpha is preferably shown as SEQ ID NO.21144-1266bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1382-422 aa.

The Cp beta is preferably 94-351 bp of a CDS sequence of an AJI77135.1 gene with an accession number, the CDS sequence of the Cp beta is preferably shown as SEQ ID NO.21267-1524bp, and the corresponding amino acid sequence is preferably shown as SEQ ID NO.1423-508 aa.

In the present invention, primers shown in Table 1 were designed based on the above sequences in the SOE-PCR:

TABLE 1 primer information for SOE-PCR

In the SOE-PCR, it is preferable to amplify the target LT Ia fragment by using the primers LT Ia 1 and LT Ia 2, amplify the target LT Ib fragment by using the primers LT Ib 1 and LT Ib 2, and amplify the target LT I fragment by using the primers LT Ib 1 and LT Ib 2; re-amplification of fragment LT I Using primers LT Ia 1 and LT I the present invention employs the same system and procedure for amplification of both the LT Ia target fragment and the LT Ib target fragment: the system is calculated by 20 mu L, and preferably comprises: 0.5 μ L of upstream primer, 0.5 μ L of downstream primer, 1 μ L of enterotoxigenic E.coli genome, 10 μ L of X5 High-Fidelity DNApolymerase PCRmix (2 ×), and H ₂ O8 mu L; said amplificationThe procedure of (2) preferably comprises 3min at 95 ℃; 30s at 95 deg.C, 30s at 63 deg.C, 1min at 72 deg.C, and 35 cycles; 5min at 72 ℃.

When primers LT Ia 1 and LT Ib 2 are used for amplifying target fragment LT I, an SOE-PCR amplification system is calculated by 20 mu L and comprises the following components: 0.5. Mu.L of the upstream primer (LT Ia 1), 0.5. Mu.L of the downstream primer (LT Ib 2), 1. Mu.L of each of the fragments LT Ia and LT Ib, 10. Mu.L of the X5 High-Fidelity DNApolymerase PCR mix (2X) and H ₂ O7 mu L; the SOE-PCR amplification program comprises: 3min at 95 ℃; 30s at 95 deg.C, 30s at 63 deg.C, 1min at 72 deg.C, and 35 cycles; and 8min at 72 ℃.

When the primers LT Ia 1 and LT I are used for amplifying the target segment LT I, the SOE-PCR amplification system is calculated by 20 mu L and comprises the following components: 0.5. Mu.L of upstream primer (LT Ia 1), 0.5. Mu.L of downstream primer (LT I), 1. Mu.L of target fragment LT I, 10. Mu.L of X5 High-Fidelity DNApolymerase PCR mix (2X) and H ₂ O8 mu L; the SOE-PCR amplification program comprises: 3min at 95 ℃; 30s at 95 deg.C, 30s at 63 deg.C, 1min at 72 deg.C, and 35 cycles; and 8min at 72 ℃.

The present invention utilizes the same system and procedure as above, utilizes primers LT II Aa1 and LT II Aa2 to amplify LT II Aa fragments by SOE-PCR, primers LT Ia 1 and LT II Aa2 to link the fragments LT I and LT II Aa into LT I-LT II Aa, and then primers LT Ia 1 and LT I-LT II Aa are used to amplify the fragments LT I-LT II Aa; the primers LT II Ab1 and LT II Ab2 are used for amplifying LT II Ab, the primers LT Ia 1 and LT II Ab2 are used for connecting the segment LT I-LT II Aa and LT II Ab into LT I-LT II Aa-LT II Ab (LT I-LT IIA), and the primers LT Ia 1 and LTI-LIIA are used for amplifying the segment LT I-LT IIA; the primers LT IIca 1 and LT IIca 2 amplify to obtain LT IIca, the primers LT Ia 1 and LT IIca 2 connect the segment LT I-LT IIA and LT IIca to form LT I-LT IIA-LT IIca, and the primers LT Ia 1 and LTI-LTIIA-LTIIca are used for amplifying the segment LT I-LT IIA-LT IIca; amplifying a segment LT IIcb by using the primers LT IIcb 1 and LT IIcb 2, connecting the segment LT I-LT IIA-LT IIca and LT IIcb by using the primers LT IIa 1 and LT IIcb 2 to form a segment LT I-LT IIA-LT IIca-LT IIcb (LT I-LT II), and amplifying the segment LT I-LT II by using the primers LT IA 1 and LTI-LTII; amplifying a segment ST a by using primers STa1 and STa2, connecting the segment LT I-LT II and ST a into a segment LT I-LT II-ST a by using the primers LT Ia 1 and LTI-LTII-STa, and amplifying the segment LT I-LT II-ST a by using the primers LT Ia 1 and LTI-LTII-STa; amplifying a segment ST b by using primers STb1 and STb2, connecting the segments LT I-LT II-ST a and ST b into a segment LT I-LT II-ST a-ST b (LT I-LT II-ST) by using the primers LT Ia 1 and LTI-LII-ST, and amplifying the segment LT I-LT II-ST by using the primers LT Ia 1 and LTI-LII-ST; amplifying a fragment Cpalpha by using primers Cpalpha 1 and Cpalpha 2, connecting the fragments LT I-LT II-ST and Cpalpha into LT I-LT II-ST-Cpalpha by using the primers LT Ia 1 and LTI-LTII-ST-Cpalpha, and amplifying the fragment LT I-LT II-ST-Cpalpha by using the primers LT Ia 1 and LTI-LTII-ST-Cpalpha; the primers Cp beta 1 and Cp beta 2 amplify the fragment Cp beta, and the primers LT Ia 1 and Cp beta 2 link the fragments LT I-LT II-ST-Cp alpha and Cp beta to form LT I-LT II-ST-Cp alpha-Cp beta (LT I-LT II-ST-Cp beta).

After obtaining the LT I-LT II-ST-Cp, the invention preferably further comprises the step of using ddH to 1 mu L of 1524bp amplification product ₂ Diluting O by 50 times, and amplifying by using primers LT Ia and Cp beta with enzyme cutting sites, wherein the amplification system is calculated by 20 mu L and comprises the following components: 1 μ L of upstream primer (LT Ia), 1 μ L of downstream primer (Cp β), 1 μ L of diluted LT I-LT II-ST-Cp, 10 μ L of 2 × Es Taq MasterMix (Dye), and 8 μ L of water. The procedure for amplification according to the invention preferably comprises: 3min at 95 ℃; 30s at 95 deg.C, 30s at 64 deg.C, 1min at 72 deg.C, and 35 cycles; and 8min at 72 ℃. The PCR product is preferably electrophoresed by using 1% agarose gel for 90V40 min, after the size of a band is verified to be correct by using a gel imaging system, a target band is cut by a knife, and a target fragment is recovered according to the operation of an agarose gel recovery kit instruction.

The recombinant expression system preferably comprises a eukaryotic expression system or a prokaryotic expression system, and more preferably comprises a prokaryotic expression system.

The invention also provides a recombinant expression vector for expressing the recombinant protein LT I-LT II-ST-Cp, wherein the basic expression vector of the recombinant expression vector comprises pET-28a, and the nucleotide sequence for coding the recombinant protein LT I-LT II-ST-Cp is inserted between the enzyme cutting sites of Nco I and Xho I of pET-28 a.

When the recombinant expression vector is constructed, the recovered target fragment is preferably connected to a pMD-18T vector, and then the connection product is transformed into an Escherichia coli E.coli DH5 alpha competent cell; after LB liquid medium culture and ampicillin screening, taking monoclonal strains for sequencing, extracting strains plasmids with correct sequencing to obtain recombinant plasmids pMD-18T-LT I-LT II-ST-Cp; the recombinant plasmid pMD-18T-LT I-LT II-ST-Cp and pET-28a vector are subjected to double enzyme digestion of Nco I and Xho I respectively and then are connected to obtain the pET-28a-LT I-LT II-ST-Cp expression vector.

The invention also provides an inducible expression method of the recombinant protein LT I-LT II-ST-Cp, which comprises the following steps: transferring the recombinant expression vector into an Escherichia coli E.coli DH5 alpha cell, extracting a plasmid, transforming Escherichia coli E.coli BL21 (DE 3), and inducing by IPTG to obtain the recombinant protein LT I-LT II-ST-Cp.

The transfer method is not particularly limited by the invention, and the conventional transfer method of the invention can be utilized. The recombinant strain with the recombinant expression vector transferred into escherichia coli cells is inoculated into a liquid LB culture medium, cultured for 2 hours at 37 ℃, added with IPTG to enable the final concentration to be 1mmol/L and induced for 8 hours.

In the invention, after the induction, the recombinant protein is preferably purified by an inclusion body purification method, and the concentration of the purified protein can be 5mg/mL.

The recombinant protein LT I-LT II-ST-Cp has good specificity of each section, no toxicity after mice are immunized, the antibody titer is higher, the antiserum has good specificity to ETEC natural enterotoxin protein and natural alpha and beta toxins of clostridium welchii, and has good protection effect on piglet diarrhea and good immune effect of 100 mu g, so the recombinant protein LT I-LT II-ST-Cp can be used for preparing a piglet diarrhea multi-vaccine.

The invention also provides application of the recombinant protein LT I-LT II-ST-Cp or the recombinant protein LT I-LT II-ST-Cp obtained by the induced expression method in preparation of a piglet diarrhea multi-linked vaccine.

The immunoadjuvant of the present invention preferably comprises an aluminum hydroxide sol adjuvant. The concentration of the recombinant protein LT I-LT II-ST-Cp in the vaccine is preferably 1 mu g/mu l, and the volume ratio of the recombinant protein LT I-LT II-ST-Cp to an immunologic adjuvant is preferably 1. The method for preparing the vaccine is not particularly limited, and the conventional vaccine preparation method in the field can be used.

The protective rate of the vaccine of the invention on the red dysentery and yellow dysentery of piglets can reach more than 80 percent, the effect is good, and the protective power is very strong.

The construction method, the expression system and the application of the concatameric fusion recombinant protein capable of preventing diarrhea of piglets provided by the invention are described in detail below with reference to the examples, but the construction method, the expression system and the application are not to be construed as limiting the scope of the invention.

In the embodiment of the invention, graphPad Prism7 statistical software is adopted to perform one-way ANOVA analysis on the experimental results of each group, and the results are expressed by Mean +/-standard deviation (Mean +/-SD). * p <0.05 was significantly different, p <0.01 was significantly different, and p > 0.05 was not significantly different.

Example 1

1. The amino acid sequence of Escherichia coli heat-labile enterotoxin LT Ia (BAA 25725.1) and the sequence of LT Ib (BAA 25726.1), LT II Aa (WP _ 095374651.1) and LT II Ab (WP _ 095389218.1), LT II ca (ALO 79807.1) and LT II cb (ALO 79813.1), heat-tolerant enterotoxin ST a (CAD 87828.1) and ST b (CAD 87835.1) and the related information of alpha toxin (AQN 80672.1) and beta toxin (AJI 77135.1) of Clostridium welchii are searched at NCBI and are subjected to antigenic analysis by DNAstar respectively.

According to the amino acid sequences of alpha toxin and beta toxin of enterotoxigenic Escherichia coli LT Ia, LT Ib, LT IIca, LT IIcb, ST a, ST b and Clostridium welchii, the antigenicity of the alpha toxin and the beta toxin is predicted by using DNA STAR, and a region with strong antigenicity is selected.

The protein structure prediction analysis is carried out to select 125 th-210 th amino acid residues of LT Ia, 74 th-115 th amino acid residues of LT Ib, 182 th-263 th amino acid residues of LT IIAa, 72 th-113 th amino acid residues of LT IIAb, 20 th-50 th amino acid residues of LT IIca, 64 th-90 th amino acid residues of LT IIcb, 20 th-58 th amino acid residues of ST a, 24 th-56 th amino acid residues of ST b, 50 th-90 th amino acid residues of clostridium welchii alpha toxin and 32 th-117 th amino acid residues of clostridium welchii beta toxin.

2. The primer set in Table 1 was designed based on the above residues and synthesized by bioengineering company.

3. Extracting the genomes of enterotoxigenic Escherichia coli and Clostridium welchii by using a genome extraction kit (Tiangen biochemistry).

4. SOE-PCR ligation of LT, ST and Clostridium welchii alpha and beta toxins

1) The target fragment LT Ia is amplified by using primers LT Ia 1 and LT Ia 2, the target fragment LT Ib is amplified by using LT Ib 1 and LT Ib 2, the target fragment LT I is connected by using primers LT Ib 1 and LT Ib 2, and the target fragment LT I is amplified by using primers LT Ib 1 and LT I.

When amplifying LT Ia and LT Ib target fragments, the system and procedure are the same:

20 μ L system: upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L, enterotoxigenic E.coli genome 1. Mu.L, X5 High-Fidelity DNA Polymerase PCR mix (2X) 10. Mu.L and H ₂ O 8μL；

And (3) amplification procedure: 3min at 95 ℃; 30s at 95 deg.C, 30s at 63 deg.C, 1min at 72 deg.C, and 35 cycles; 5min at 72 ℃.

Preparing 20 mu.L SOE-PCR system, and amplifying a target fragment LT I by using primers LT I1 and LT Ib 2: 0.5. Mu.L of the upstream primer (LT Ia 1), 0.5. Mu.L of the downstream primer (LT Ib 2), 1. Mu.L of each of LT Ia and LT Ib, 10. Mu.L of the X5 High-Fidelity DNA Polymerase PCR mix (2X) and H ₂ O7 mu L; the procedure is as follows: 3min at 95 ℃; 30s at 95 ℃, 30s at 63 ℃, 1min at 72 ℃ and 35 cycles; and 8min at 72 ℃.

Preparing 20 mu.L of SOE-PCR system, amplifying target fragment LT I by using primers LT Ia 1 and LT I: 0.5. Mu.L of forward primer (LT Ia 1), 0.5. Mu.L of reverse primer (LT I), 1. Mu.L of target fragment LT I, 10. Mu.L of X5 High-Fidelity DNA Polymerase PCR mix (2X) and H ₂ O8 mu L; the SOE-PCR amplification program comprises: 3min at 95 ℃; 30s at 95 ℃, 30s at 63 ℃, 1min at 72 ℃ and 35 cycles; and 8min at 72 ℃.

2) The invention utilizes the same system and program as above, utilizes the primers LT II Aa1 and LT II Aa2 to amplify the LT II Aa segment by SOE-PCR, the primers LT Ia 1 and LT II Aa2 connect the segment LT I and LT II Aa into LT I-LT II Aa, and then the primers LT Ia 1 and LTI-LTIIAa are used to amplify the segment LT I-LT II Aa; the primers LT II Ab1 and LT II Ab2 are used for amplifying LT II Ab, the primers LT Ia 1 and LT II Ab2 are used for connecting the segment LT I-LT II Aa and LT II Ab into LT I-LT II Aa-LT II Ab (LT I-LT IIA), and the primers LT Ia 1 and LTI-LIIA are used for amplifying the segment LT I-LT IIA; the primers LT IIca 1 and LT IIca 2 amplify to obtain LT IIca, the primers LT Ia 1 and LT IIca 2 connect the segment LT I-LT IIA and LT IIca to form LT I-LT IIA-LT IIca, and the primers LT Ia 1 and LTI-LTIIA-LTIIca are used for amplifying the segment LT I-LT IIA-LT IIca; the primers LT IIcb 1 and LT IIcb 2 amplify a segment LT IIcb, the primers LT Ia 1 and LT IIcb 2 connect the segment LT I-LT IIA-LT IIca and LT IIcb into a segment LT I-LT IIA-LT IIca-LT IIcb (LT I-LT II), and the primers LT Ia 1 and LT I-LTII are used for amplifying a segment LT I-LT II; amplifying a segment ST a by using primers STa1 and STa2, connecting the segment LT I-LT II and ST a into a segment LT I-LT II-ST a by using the primers LT Ia 1 and LTI-LTII-STa, and amplifying the segment LT I-LT II-ST a by using the primers LT Ia 1 and LTI-LTII-STa; amplifying a segment ST b by using primers STb1 and STb2, connecting the segments LT I-LT II-ST a and ST b into a segment LT I-LT II-ST a-ST b (LT I-LT II-ST) by using the primers LT Ia 1 and LTI-LII-ST, and amplifying the segment LT I-LT II-ST by using the primers LT Ia 1 and LTI-LII-ST; amplifying a fragment Cpalpha by using primers Cpalpha 1 and Cpalpha 2, connecting the fragments LT I-LT II-ST and Cpalpha into LT I-LT II-ST-Cpalpha by using the primers LT Ia 1 and LTI-LTII-ST-Cpalpha, and amplifying the fragment LT I-LT II-ST-Cpalpha by using the primers LT Ia 1 and LTI-LTII-ST-Cpalpha; the fragments Cp β are amplified by primers Cp β 1 and Cp β 2, and the fragments LT I-LT II-ST-Cp α and Cp β are ligated by primers LT Ia 1 and Cp β to LT I-LT II-ST-Cp α -Cp β (LT I-LT II-ST-Cp). (FIG. 1).

3) 1 μ L of 1524bp amplification product was treated with ddH ₂ O was diluted 50-fold and amplified with primers LT Ia and Cp β with restriction sites, system (20. Mu.L): 1 μ L of upstream primer (LT Ia), 1 μ L of downstream primer (Cp β), 1 μ L of diluted LT I-LT II-ST-Cp, 10 μ L of 2 × Es Taq MasterMix (Dye), and 8 μ L of water.

And (3) amplification procedure: 3min at 95 ℃; 30s at 95 deg.C, 30s at 64 deg.C, 1min at 72 deg.C, and 35 cycles; and 8min at 72 ℃.

And (3) carrying out electrophoresis on the PCR product by using 1% agarose gel, 90V 40min, verifying the correct size of the strip by using a gel imaging system, cutting down the target strip by using a small knife, and recovering the target fragment according to the operation of an agarose gel recovery kit (Tiangen biochemistry) instruction.

5. Construction of cloning vectors

1) Connecting the target fragment verified and recovered by PCR to a pMD-18T vector;

2) Mixing the ligation product with E.coli DH5 alpha competent cells, and standing on ice for 30min;

3) Immediately transferring to 42 ℃ for heat shock for 90s, and then immediately putting on ice for cold shock for 10min;

4) Adding 400 μ L of nonresistant LB liquid medium, and culturing in a shaker at 37 deg.C for 90min;

5) Taking 100 mu L of the bacterial liquid in the step 4), coating the bacterial liquid on an LB solid plate containing ampicillin, and culturing at the constant temperature of 37 ℃ for 8h;

6) Selecting a monoclonal strain, inoculating the strain into an LB liquid culture medium containing ampicillin, culturing at a constant temperature of 37 ℃ for 8 hours, and sequencing;

7) The correctly sequenced strains were cryopreserved and plasmids were extracted.

6. Construction of expression plasmids

1) The recombinant plasmid pMD-18T-LT I-LT II-ST-Cp and the empty vector pET-28a are subjected to double enzyme digestion respectively, and the enzyme digestion system is 30 mu L: recombinant plasmid/empty vector 24.0. Mu.L, nco I1.5. Mu.L, xho I1.5. Mu.L and 10 XKbuffer 3.0. Mu.L; acting for 6 hours at 37 ℃;

2) Performing 1% agarose gel electrophoresis on the double digestion product, and performing gel recovery on the LT I-LT II-ST-Cp target fragment and pET-28 a;

3) Connecting the LT I-LT II-ST-Cp target fragment recovered from the gel after enzyme digestion obtained in the step 2) with the pET-28a carrier fragment to construct a pET-28a-LT I-LT II-ST-Cp expression carrier;

ligation reaction (10 μ L): 2 muL of LT I-LT II-ST-Cp, 3.5 muL of double enzyme digestion pET-28a carrier and 4.5 muL of Solution I; reacting for 8 hours at the temperature of 16 ℃;

4) Transferring the ligation product in 3) into E.coli DH5 alpha according to the method;

5) Selecting a monoclonal strain, culturing and sequencing;

6) Extracting plasmids from strains with correct sequencing, and operating according to the specification of a small plasmid extraction kit of Tiangen;

7) And transforming the extracted recombinant plasmid pET-28a-LT I-LT II-ST-Cp into E.coli BL21 (DE 3) competence according to the same operation, selecting bacteria, culturing, sequencing, and identifying the correct strain for conservation.

7. Inducible expression of recombinant proteins

1) Induction: inoculating the E.coli BL21 (DE 3) strain into a liquid LB culture medium, culturing at 37 ℃ for 2h, adding IPTG (isopropyl-beta-D-thiogalactoside) to enable the final concentration to be 1mmol/L, inducing for 8h, and simultaneously performing no-load negative control;

2) Preparing a sample: centrifuging and collecting induced and non-induced bacteria liquid with the same bacteria amount, re-suspending with PBS, adding SDS loading buffer solution, and boiling for 10min;

3) Performing SDS-PAGE electrophoresis, and loading 7 mu L of Marker sample to 10 mu L of sample to be detected in each hole; carrying out electrophoresis on 5% of lamination glue for 30min by using a 90V voltage, and carrying out electrophoresis on 12% of separation glue for 1.5h by using a 120V voltage;

4) Dyeing and decoloring: and (3) dyeing the separation gel subjected to electrophoresis for 4 hours at room temperature by using newly prepared Coomassie brilliant blue R-250, decoloring for 8 hours, changing the decoloring solution every 2 hours, and collecting an image by using a gel imaging system after the completion.

As shown in FIG. 2, the constructed recombinant expression strain was analyzed by IPTG induction at 37 ℃ and SDS-PAGE electrophoresis. The results show that successful expression of the recombinant LT I-LT II-ST-Cp protein has a distinct band at 55kDa, which is the same as the theoretical size.

Example 2

Purification and activity verification of LT I-LT II-ST-Cp recombinant protein

1. Protein purification

1) Recombinant E.coli BL21 (DE 3) strain of LT I-LT II-ST-Cp was inoculated in an amount of 100. Mu.L into 5ml of liquid LB, then one in a thousand parts of kanamycin was added, and shake-cultured at 37 ℃ and 160rpm for 3 hours.

2) And (2) inoculating 5ml of the bacterial liquid obtained in the step (1) into 1L of sterilized LB, adding one thousandth of kanamycin, culturing for 2h by using a shaking table at 37 ℃ and 160rpm, adding IPTG (isopropyl-beta-thiogalactoside) to enable the final concentration to be 1mmol/L, and inducing for 24h by using a shaking table at 16 ℃ and 160 rpm.

3) And (3) centrifuging the bacterial liquid obtained in the step (2) at 8000rpm for 10min at 4 ℃, collecting the bacteria, performing PBS ultrasonic crushing, performing ice ultrasonic crushing after performing ice bath for 10min, performing ultrasonic crushing at intervals of 3s for 30min, centrifuging at 8000rpm for 10min at 4 ℃, discarding supernatant, and collecting precipitates. Washed 3 times with PBS and centrifuged as before.

4) And (4) taking the precipitate obtained in the previous step, weighing the wet weight, and suspending the wet weight by using PBS to a required concentration, and storing the wet weight at-80 ℃ for later use.

The results are shown in fig. 3, after the recombinant protein is successfully expressed, the recombinant protein is purified by an inclusion body purification method, so that the target band is clear and single, the size is consistent, the purification effect is better, and the concentration of the purified protein is 5mg/mL.

2. Recombinant protein specificity verification

2.1 validation of recombinant proteins with commercial anti-LT Ia antibodies

1) Electrophoresis: performing SDS electrophoresis on the sample, and activating the PVDF membrane by using methanol after the SDS electrophoresis is finished;

2) Film transferring: the film is rotated by adopting a wet rotation method, and the assembly sequence from the negative electrode to the positive electrode is as follows: filter paper/glue/membrane/filter paper, in the membrane transfer buffer solution in the whole process, preventing bubbles from generating between every two layers, wherein the membrane transfer condition is 200mA,2h;

3) And (3) sealing: sealing overnight at 4 ℃ by using sealing liquid after the membrane transfer is finished;

4) A first antibody: diluting LT antibody with 2000 times of blocking solution, incubating at 37 deg.C for 1h, washing with PBST for three times for 15 min/time;

5) Secondary antibody: diluting goat anti-mouse IgG marked by HRP (horse radish peroxidase) by 4000 times by using a confining liquid, incubating for 1h at 37 ℃, washing for three times by PBST (para-phenylene benzobisoxazole) (15 min/time);

6) Adding ECL color developing solution and collecting image information by an imager.

7) The western activity test was performed using commercially available antibodies against the alpha toxin and beta toxin of LT Ib, LT IIAa, LT IIAb, LT IIca, LT IIcb, ST a, ST b, clostridium welchii, respectively, in the same manner as above.

As a result, as shown in FIG. 4, the specificity of each fragment of the recombinant protein was good.

2.2 toxicity verification

1) Balb/c mice were immunized with purified and unpurified recombinant protein, 5 mice per group, 100. Mu.g/mouse. Wherein the unpurified group is not subjected to ultrasonic disruption after induction, is centrifuged at 8000rpm for 10min at 4 ℃, is discarded, is weighed to be wet, is directly resuspended to the required concentration by formaldehyde with the final concentration of 3 percent, and is inactivated for 24h.

2) In the period, the mouse culture cage is not filled with padding, the state of the mouse is observed every 3h, whether the hair is upright or disordered, and whether stress symptoms such as diarrhea or vomiting exist.

After three immunizations of mice, the mice of the purified and unpurified recombinant protein groups were normal in state, smooth in fur, free from diarrhea and vomiting symptoms, and free from vomit in cages after each immunization, indicating that the recombinant protein was inactive.

2.3 detection of the Change Pattern of the antibody

1) The immunization schedule shown in table 2 was prepared, wherein the first immunization was emulsified with equal volume of freund's complete adjuvant and the subsequent immunization was emulsified with freund's incomplete adjuvant for 14 days.

TABLE 2 immunization procedure

	Purification of Low doses	Purifying high dose	Low dose without purification	High dose without purification	0.01MPBS
						One need not	30μg	100μg	30μg	100μg	Same volume (100 mu L)
Two exempt from	30μg	100μg	30μg	100μg	Same volume (100 mu L)
						Sanwu (Chinese character of 'Sanwu')	30μg	100μg	30μg	100μg	Same volume (100 mu L)

2) Blood was collected from the tip of each mouse on

days

3, 7 and 11 of each immunization, the blood was placed at 37 ℃ for 30min, centrifuged at 2000rpm in a centrifuge at 4 ℃ for 20min, and the supernatant was slowly pipetted into a clean tube.

3) The purified recombinant protein of LT I-LT II-ST-Cp was coated on ELISA plates at a concentration of 1 ng/. Mu.L, 100. Mu.L/well, and incubated at 37 ℃ in an incubator for 2h.

4) The coated ELISA wells were blocked with 5% skim milk powder at 50. Mu.L/well and incubated at 37 ℃ for 1h.

5) PBST was washed three times, each time at 1min, 200. Mu.L/well.

6) Serum from each mouse in step 2 was diluted from 250 fold with PBS multiple as primary antibody, 100. Mu.L/well, added to the wells of

step

5, 3 replicates per well, and incubated at 37 ℃ in an incubator for 1h.

7) PBST was washed three times, each time at 1min, 200. Mu.L/well.

8) Diluting goat anti-mouse IgG by 4000 times with PBS, adding 100 microliters of the diluted goat anti-mouse IgG into the treated wells in the step 7), and incubating for 1h at 37 ℃ in an incubator.

9) PBST was washed three times, each time at 1min, 200. Mu.L/well.

10 Adding a TMB substrate developing solution into the wells after the treatment of the step 9), incubating at 100 mu L/well and keeping away from light at 37 ℃ for 8min.

11 Add stop solution, 50. Mu.L/well, to the TMB substrate in step 10), keep each well free of macroscopic bubbles, and read the OD at 450nm with a microplate reader.

The results are shown in figure 5, with highest serum antibody levels at day 7 after each immunization, with highest mouse serum titers in the purified and unpurified 100 μ g groups.

2.4 specificity validation of antisera

Blood is collected from the eyeballs of the mice 14 days after the group of 100 mu g purified three-immunity, 2000-fold dilution of serum is collected to be used as a primary antibody, a western experiment is carried out by taking ETEC and clostridium welchii as solid phase antigens, the specificity of the antiserum is verified, and the specific operation steps are the same as the above.

As shown in fig. 6 and fig. 7, the specificity of ETEC-verified antiserum showed two bands, 85KDa is LT toxin, 16KDa is ST toxin, and antiserum can bind to native protein ETEC enterotoxin with good specificity (fig. 6); the figure for the verification of antiserum specificity by clostridium welchii shows that two bands, namely alpha toxin and beta 1 toxin in the sequence of 43KDa and 37KDa, show that antiserum can be combined with alpha and beta 1 toxin of natural clostridium welchii, and the specificity is good (figure 7).

2.5 Immunopotentiality test

1) Mice were immunized according to the protocol of Table 2, but 10 balb/c mice were placed in each group.

2) Preparing LB culture medium in advance, inoculating 100 μ L each of Clostridium welchii and enterotoxigenic Escherichia coli, culturing at 37 deg.C and 160rpm for 8 hr.

3) Day 14 after the third immunizationThe challenge experiment was carried out on mice, each mouse was challenged with 1.5X 10 ⁷ The toxin counteracting method is intraperitoneal injection and observation for 10 days.

As shown in Table 3, when the recombinant protein LT I-LT II-ST-Cp was purified, the protection rate of the 30. Mu.g group was 70% and the protection rate of the 100. Mu.g group was 90%, and when the recombinant protein was not purified, the protection rate of the 30. Mu.g group was 80% and the protection rate of the 100. Mu.g group was 100%, all the control groups were attacked. Therefore, the recombinant protein LT I-LT II-ST-Cp has good protection effect on diarrhea and good immune effect of 100 mu g, wherein the unpurified group is superior to the purified group.

TABLE 3 Immunopotential test results

Grouping	Laboratory mouse (one)	Number of onset (number)	Death number (one)	Protective Rate (%)
					Purification of 30. Mu.g	10	3	1	70
Purification 100. Mu.g	10	1	0	90
					Without purification 30. Mu.g	10	2	1	80
Not purified 100. Mu.g	10	0	0	100
					PBS	10	10	8	0

Example 3

Immunoprotective experiment of recombinant protein LT I-LT II-ST-Cp on piglets

1. Laboratory strains and animals

The newly born long white pigs are purchased from a Changchun pig farm (piglet diarrhea never occurs in the pig farm and no vaccine is inoculated to the piglets after birth), 50 piglets are purchased and are raised in a standard pigsty, the piglets are raised according to the standard by professionals, each pig is independently closed to be raised, and no antibiotic is added into the feed.

2. Preparation of vaccines

The recombinant protein was diluted to 1 μ g/μ l, and the aluminum hydroxide sol adjuvant was slowly added to the recombinant protein (1 g adjuvant per piglet) in a volume ratio of 1.

3、

3.1 Vaccination and immunization

1) Experiment grouping

40 piglets were randomly divided into 4 groups, and 10 piglets per group were immunized according to the following immunization program with an immunization cycle of 14 days in a neck intramuscular injection manner.

TABLE 4 Experimental groups

	Purification group	Non-purified fraction	0.01MPBS	0.01MPBS + adjuvant
					One free	1mg	1mg	Same volume (2 ml)	Same volume (2 ml)
Secondary drug	1mg	1mg	Same volume (2 ml)	Same volume (2 ml)
					Sanwu	1mg	1mg	Same volume (2 ml)	Same volume (2 ml)
Exempt from	1mg	1mg	Same volume (2 ml)	Same volume (2 ml)

2) On the 7 th day after the four-immunization, each group of gavage infected enterotoxigenic Escherichia coli and Clostridium welchii were gavaged with 2X 10 per pig ⁸ ETEC and Clostridium welchii, the drenching volume is 5ml.

3) After infection with ETEC and clostridium welchii, the status of the piglets was observed every 4h, as to whether the hair was rough, whether the body temperature was normal, whether the faeces were yellow or red thin paste, whether the piglets were anorexia, unwilling, whether they were emaciable (which may be weighed), and whether there was death.

4) And counting the number of the piglets died of each group 14 days after bacteria attack.

The results are shown in table 5, and the protection rate of the piglet immunized by the recombinant protein as a vaccine can reach more than 80%, and the effect is good. The sick piglets are in good mental state, the color and the shape of the excrement are normal, and the body temperature and the weight are normal; the hair of the sick piglets is dull and disordered, the body temperature is raised, the body is thin, the excrement is red and yellow thin paste, and the whole mental state is more cachectic and listless.

TABLE 5 statistical table of piglet morbidity and status

Grouping	Number of laboratory animals	Number of animals affected(A)	Number of dead animals	Protective Rate (%)
					Purification group	10	2	0	80
Non-purified fraction	10	1	0	90
					0.01MPBS	10	9	7	10
0.01MPBS + adjuvant	10	8	6	20

3.2 piglet intestinal State Observation

The experimental piglets (including the sick and dead piglets) are killed 10 days after the attack of the bacteria, dissected and observed whether the intestinal state of the experimental piglets has pathological changes or not.

The results show that the intestinal tracts of the piglets which are not attacked have no obvious pathological changes and are relatively normal. The intestinal mucosa of the sick piglet has hyperemia, necrosis and desquamation, intestinal swelling, blood sample or yellow liquid filled in the content, mesenteric lymph node hyperemia and swelling, and the intestinal mucosa has acute catarrhal inflammation. The anus is loose and is purple red, the tail part is stained with feces, and the feces are red or yellow and thin paste. The spleen of all the sick or dead piglets is obviously enlarged and has bleeding points and dead spots.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Weifang Xishan Weitai Biotech limited

<120> construction method, expression system and application of multi-union fusion recombinant protein capable of preventing piglet diarrhea

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 508

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

His Pro Tyr Glu Gln Glu Val Ser Ala Leu Gly Gly Ile Pro Tyr Ser

1 5 10 15

Gln Ile Tyr Gly Trp Tyr Arg Val Asn Phe Gly Val Ile Asp Glu Arg

20 25 30

Leu His Arg Asn Arg Glu Tyr Arg Asp Arg Tyr Tyr Arg Asn Leu Asn

35 40 45

Ile Ala Pro Ala Glu Asp Gly Tyr Arg Leu Ala Gly Phe Pro Pro Asp

50 55 60

His Gln Ala Trp Arg Glu Glu Pro Trp Ile His His Ala Pro Gln Gly

65 70 75 80

Cys Gly Asn Ser Ser Arg Gly Ser Gln His Ile Asp Ser Gln Lys Lys

85 90 95

Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr Tyr Leu Thr Glu

100 105 110

Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn Ala

115 120 125

Gly Phe Pro Ala Asn Phe Pro Ala Trp Arg Glu Thr Pro Trp Ser Thr

130 135 140

Phe Ala Pro Glu Gln Cys Gly Pro Asn Lys Lys Thr Ile Lys Glu Leu

145 150 155 160

Ser Cys Thr Ser Ala Thr Asn Ala Leu Ser Lys Tyr Asp Leu Met Asn

165 170 175

Phe Lys Lys Phe Leu Lys Arg Arg Leu Thr Leu Met Phe Leu Ile Ser

180 185 190

Glu Asp Asp Phe Ile Asn Val His Gly Asp Arg Asn Glu Phe Tyr Lys

195 200 205

Asp Ile Pro Gly Gly Arg Asp Tyr Pro Asp Asn Ile Leu Ala Ser Glu

210 215 220

Ile Arg Arg Ile Ala Val Ala Ala Ile Leu Ser Asp Ala Lys Val Asn

225 230 235 240

Leu Cys Ala Lys Thr Ser Ser Ser Pro Asn His Asp Phe Phe Arg Ala

245 250 255

Asp Ser Arg Thr Pro Asp Glu Ile Arg Arg Ala Gly Gly Leu Leu Pro

260 265 270

Arg Gly Gln Gln Glu Ala Tyr Glu Arg Gly Asp Ser Thr Gly Lys Thr

275 280 285

Arg Phe Ile Pro Gly Gly Gln Tyr Tyr Pro Glu Asn Tyr Leu Ser Asn

290 295 300

Glu Met Arg Lys Ile Gln Ser Thr Glu Ser Leu Asp Ser Ser Lys Glu

305 310 315 320

Lys Ile Thr Leu Glu Thr Lys Lys Cys Asp Val Val Lys Asn Asn Ser

325 330 335

Glu Lys Lys Ser Glu Asn Met Asn Asn Thr Phe Tyr Ser Thr Gln Ser

340 345 350

Asn Lys Lys Asp Leu Cys Glu Asn Tyr Arg Gln Ile Ala Lys Glu Ser

355 360 365

Cys Lys Ile Gly Phe Leu Gly Val Arg Asp Gly Thr Ala Leu Glu Asn

370 375 380

Asp Leu Ser Lys Asn Glu Pro Glu Ser Val Arg Lys Asn Leu Glu Ile

385 390 395 400

Leu Lys Glu Asn Met His Glu Leu Gln Leu Gly Ser Thr Tyr Pro Asp

405 410 415

Tyr Asp Lys Asn Ala Tyr Lys Thr Thr Thr Ile Thr Arg Asn Lys Thr

420 425 430

Ser Asp Gly Tyr Thr Ile Ile Thr Gln Asn Asp Lys Gln Ile Ile Ser

435 440 445

Tyr Gln Ser Val Asp Ser Ser Ser Lys Asn Glu Asp Gly Phe Thr Ala

450 455 460

Ser Ile Asp Ala Arg Phe Ile Asp Asp Lys Tyr Ser Ser Glu Met Thr

465 470 475 480

Thr Leu Ile Asn Leu Thr Gly Phe Met Ser Ser Lys Lys Glu Asp Val

485 490 495

Ile Lys Lys Tyr Asn Leu His Asp Val Thr Asn Ser

500 505

<210> 2

<211> 1524

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

cacccatatg aacaggaggt ttctgcgtta ggtggaatac catattctca gatatatgga 60

tggtatcgtg ttaattttgg tgtaattgat gaacgattac atcgtaacag ggaatataga 120

gaccggtatt acagaaatct gaatatagct ccggcagagg atggttacag attagcaggt 180

ttcccaccgg atcaccaagc ttggagagaa gaaccctgga ttcatcatgc accacaaggt 240

tgtggaaatt catcaagagg cagtcaacat atagactccc aaaaaaaagc cattgaaagg 300

atgaaggaca cattaagaat cacatatctg accgagacca aaattgataa attatgtgta 360

tggaataata aaacccccaa tgctgggttc cctgctaatt tcccagcatg gagagaaacg 420

ccatggagca catttgctcc tgaacagtgt ggtccgaata aaaaaacaat taaagagtta 480

tcgtgcactt ctgctacaaa tgcattatca aaatatgact taatgaattt caaaaagttt 540

ttaaagcgca ggttgacttt aatgttttta ataagtgaag acgattttat taacgtgcat 600

ggagacagaa atgaattcta caaagatatt cctggtggtc gggattatcc ggataatatc 660

ctggctagtg aaatcagaag aattgcagtg gcagccattc tctcagacgc aaaggtaaat 720

ttgtgcgcta agacttcttc tagtcctaat catgatttct ttagagcaga ctccagaaca 780

ccagatgaaa taagacgtgc gggagggctt ttaccaagag ggcagcagga ggcttatgag 840

cgcggagatt ctacaggaaa aaccagattc attcctgggg ggcagtacta tcccgagaat 900

tatctgagca atgagatgag gaaaatacag tcaactgaat cacttgactc ttcaaaagag 960

aaaattacat tagagactaa aaagtgtgat gttgtaaaaa acaacagtga aaaaaaatca 1020

gaaaatatga acaacacatt ttactctaca caatcaaata aaaaagatct gtgtgaaaat 1080

tatagacaaa tagccaagga aagttgtaaa ataggttttt taggggttag agatggtact 1140

gctttagaaa atgatctgtc caaaaatgaa ccagaaagtg taagaaaaaa cttagagatt 1200

ttaaaagaga acatgcatga gcttcaatta ggttctactt atccagatta tgataagaat 1260

gcatataaaa ctactactat aactagaaat aagacatcag atggctatac tataattaca 1320

caaaatgata aacagataat atcatatcaa tctgttgact cttcaagtaa aaatgaagat 1380

ggttttactg catctataga tgctagattt atcgatgata aatattcatc tgaaatgaca 1440

actttaataa acttaactgg atttatgtct tcaaaaaaag aagatgttat aaaaaaatac 1500

aatttgcatg atgttactaa ttct 1524

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

cacccatatg aacaggaggt t 21

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gagtctatat gttgactgcc tcttgatgaa tttccacaac 40

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

gttgtggaaa ttcatcaaga ggcagtcaac atatagactc 40

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

attgggggtt ttattattcc a 21

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

aaattagcag ggaacccagc attgggggtt ttattattcc 40

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ggaataataa aacccccaat gctgggttcc ctgctaattt 40

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

atctttgtag aattcatttc 20

<210> 10

<211> 41

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

ataatcccga ccaccaggaa tgaaatgaat tctacaaaga t 41

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

atctttgtag aattcatttc attcctggtg gtcgggatta t 41

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

atgattagga ctagaagaag 20

<210> 13

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

tggagtctgc tctaaagaaa tcatgattag gactagaaga ag 42

<210> 14

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

cttcttctag tcctaatcat gatttcttta gagcagactc ca 42

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 15

tccgcgctca taagcctcct 20

<210> 16

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

ctggtttttc ctgtagaatc tccgcgctca taagcctcct gc 42

<210> 17

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

gcaggaggct tatgagcgcg gagattctac aggaaaaacc ag 42

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

gcaatgagat gaggaaaata 20

<210> 19

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 19

caagtgattc agttgactgt attttcctca tctcattgc 39

<210> 20

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

gcaatgagat gaggaaaata cagtcaactg aatcacttg 39

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 21

gtaaaatgtg ttgttcatat 20

<210> 22

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 22

tttttatttg attgtgtaga gtaaaatgtg ttgttcatat 40

<210> 23

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 23

atatgaacaa cacattttac tctacacaat caaataaaaa 40

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 24

agcagtacca tctctaaccc 20

<210> 25

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 25

tggacagatc attttctaaa gcagtaccat ctctaacccc 40

<210> 26

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 26

ggggttagag atggtactgc tttagaaaat gatctgtcca 40

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 27

atatgcattc ttatcataat c 21

<210> 28

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 28

tagttatagt agtagtttta tatgcattct tatcataatc 40

<210> 29

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 29

gattatgata agaatgcata taaaactact actataacta 40

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 30

agaattagta acatcatgca 20

<210> 31

<211> 31

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 31

cgccatggca cacccatatg aacaggaggt t 31

<210> 32

<211> 28

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 32

gcctcgagag aattagtaac atcatgca 28

Claims

1. The concatameric fusion recombinant protein LT I-LT II-ST-Cp is characterized in that the amino acid sequence of the recombinant protein LT I-LT II-ST-Cp is shown as SEQ ID No. 1.

2. The recombinant protein LT I-LT II-ST-Cp as claimed in claim 1, characterized in that the nucleotide sequence coding for the recombinant protein LT I-LT II-ST-Cp is shown in SEQ ID No. 2.

3. A method for constructing a gene coding for the recombinant protein LT i-LT ii-ST-Cp according to claim 1 or 2, comprising the steps of: and (3) connecting a partial functional region of a heat-resistant enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of a thermolabile enterotoxin gene derived from enterotoxigenic escherichia coli, a partial functional region of an alpha toxin gene derived from clostridium welchii and a partial functional region of a beta 1 toxin gene derived from clostridium welchii in series by using an SOE-PCR method to obtain the coding gene.

4. The method of claim 3, wherein the functional regions of the heat-labile enterotoxin gene derived from enterotoxin-producing Escherichia coli include: LT Ia, LT Ib, LT IIAa, LT II Ab, LT II ca and LT II cb; the CDS sequence of LT Ia is shown as 1-258bp of SEQ ID NO.2, the CDS sequence of LT Ib is shown as 259-381bp of SEQ ID NO.2, the CDS sequence of LT II Aa is shown as 382-627bp of SEQ ID NO.2, the CDS sequence of LT II Ab is shown as 628-753bp of SEQ ID NO.2, the CDS sequence of LT II ca is shown as 754-846bp of SEQ ID NO.2, and the CDS sequence of LT II cb is shown as 847-927bp of SEQ ID NO. 2;

the partial functional region of the heat-resistant enterotoxin gene derived from enterotoxigenic escherichia coli comprises: STa and STb; the CDS sequence of the STa is shown as 928-1044bp of SEQ ID NO.2, and the CDS sequence of the STb is shown as 1045-1143bp of SEQ ID NO. 2;

5. Recombinant expression vector for the expression of the recombinant protein LT I-LT II-ST-Cp according to claim 1 or 2, characterized in that the base vector of the recombinant expression vector comprises pET-28a, the nucleotide sequence coding for the recombinant protein LT I-LT II-ST-Cp being inserted between the NcoI and XhoI cleavage sites of pET-28 a.

6. The method for inducible expression of the recombinant protein LT I-LT II-ST-Cp as defined in claim 1 or 2, comprising the steps of: transferring the recombinant expression vector of claim 5 into Escherichia coli E.coli DH5 alpha cells, extracting plasmids, transforming Escherichia coli E.coli BL21 (DE 3) competent cells, and inducing with IPTG to obtain the recombinant protein LT I-LT II-ST-Cp.

7. Use of the recombinant protein LT I-LT II-ST-Cp as defined in claim 1 or 2 or the recombinant protein LT I-LT II-ST-Cp obtained by the induction expression method as defined in claim 6 for preparing a concatenated vaccine for piglet diarrhea.

8. A piglet diarrhea concatenated subunit genetic engineering vaccine, which comprises the recombinant protein LT I-LT II-ST-Cp disclosed in claim 1 or 2 or the recombinant protein LT I-LT II-ST-Cp obtained by the induced expression method disclosed in claim 6 and an immunologic adjuvant.